Pediatric admissions to emergency departments of North-Western Italy during COVID-19 pandemic: A retrospective observational study

COVID-19 pandemic caused huge decrease of pediatric admissions to Emergency Department (ED), arising concerns about possible delays in diagnosis and treatment of severe disorders

Refinement of a macaque transplantation model: application of a subcutaneous port as a means for long-term enteral drug administration and nutritional supplementation.

A new application of a device enabling the long-term enteral administration of drugs or nutritional supplementation was developed for implementing in research entailing the use of macaques (Macaca fascicularis). After implanting a subcutaneous port, a surgically-placed gastrostomy (SPG) was completed to afford access to the gastric lumen and enable the administration of substances. In this study, the device was left in place for a period ranging between two and 12 months in macaques (n = 16). In five cases, the SPG was used successfully for 8-12 months, until the experimental endpoint was reached. In six cases, the SPG had to be removed earlier due to local infection at the implant site, which promptly regressed after the SPG was removed and antibiotic treatment was administered. One SPG-implanted macaque was euthanized for reasons unrelated to the SPG or the xenotransplantation procedure. In four cases, the SPG was implanted without any complications but has yet to be used to administer substances to the animals. From an ethical standpoint, the SPG device described here minimizes the forced handling of macaques otherwise needed for the oral administration of viscous or unpalatable substances by gavage. The device thus represents an effective refinement that fully complies with the tenet of the '3 Rs' that should be considered by primate centres exposing non-human primates to the long-term daily administration of substances by oral gavage

Isolation and characterization of porcine Gal KO fibroblasts expressing hCD55-hCD39 and hEPCR-hTPA

Xenotransplantation will benefit from genetic modification of the pig genome to reduce immunogenicity; the first obstacle in pig to human xenotransplanation was represented by hyperacute rejection (HAR), which has been overcome by genetic ablation of a1,3 galactosyltransferase and by expression of the hDAF. The second obstacle is represented by acute vascular rejection (AVR) that is characterised by vascular thrombosis, blood extravasation and edema. Expression of molecules involved in the coagulation cascade (ePCR and tPA) and in inflammatory-apoptotic events (CD39) are potential strategies to bypass AVR. The aim of this work is the production of transgenic cell lines to use in SCNT for the production of pigs for xenotransplantation studies. Two neonatal pig Gal KO fibroblasts lines cultured in DMEM/M199 1:1 + 10% FCS + 5ng/ml bFGF were co-transfected by nucleofection with ubiquitous expression vectors pMG5\u20193\u2019MARHyg2272-CXhDAF and pCXhCD39-3'MAR or pMG5\u20193\u2019MARPuro5171-CXhEPCR and pJC13I-CXhTPA. After nucleofection, cells were plated in Petri dishes and selected for eight days with Hygromycin: 150 \ub5g/ml or Puromycin: 1 \ub5g/ml. Drug resistant colonies were isolated and expanded for transgene expression analysis. We used immunohistochemistry (IHC) to detect the expression of the protein. For hDAF we used IA10 (BD Pharmingen) and for hCD39 BU61(Ancell). For hEPCR and hTPA we screened a battery of antibodies without detecting a clear specific signal. Therefore we used RT-PCR to detect the presence of the transcripts. Cells from DAF-CD39 colonies were serum starved for 24h before being fused to enucleated oocytes. Following electric activation, embryos were grown in vitro to the blastocyst stage. Thirty-nine colonies derived from a cell line transfected with hDAF-hCD39 were analysed by IHC: 48,7 % of the colonies expressed both hCD55-hCD39; 28,2 % expressed only CD55; 15,3 % expressed only CD39; 5,1 % showed mosaic expression. IHC findings were confirmed also by RT PCR. We analysed nine selected colonies from hEPCR-hTPA by RT PCR; six colonies (66.6 %) showed the presence of both transcripts, two colonies (22.2 %) showed only TPA transcript. Gal KO cell colonies co-expressing DAF-CD39 were used in SCNT experiments obtaining 32.4% compacted blastocyst development (n=1446). We obtained 40.7 % (n=2583) blastocyst development using only Gal KO cells. In this experiment was demonstrated that this system is very efficient to produce DAF-CD39 cloned pig blastocysts using GAL KO cells. Moreover, Gal KO TPA/EPCR cell lines were established for the first time. This study was supported by EU grant n_ LSHB-CT-2006-037377 and Fondazione Banca Popolare di Cremon

Quantitative and qualitative evaluation of proteinuria in non human primates recipients of porcine xenografts

Introduction: Immunological and histopathological features in pig-to-primate renal xenotransplantation are widely studied. However, only limited data have been reported on clinical-pathological findings in primate recipients of life supporting renal xenografts. In human medicine, proteinuria represents a common complication in kidney transplantation and is associated with impaired patient and graft survival. The detection of low molecular weight proteins of tubular origin such as \u3b11-microglobulin, \u3b22-microglobulin, retinol-binding protein, lysozime, is considered an early marker for predicting potential graft rejection. In the present study the presence and significance of quantitative and qualitative proteinuria were evaluated in xenotransplanted non-human primates in which kidney function is supported only by the transplanted organ. Material and methods: Eight captive-bred, bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis), recipient of a life-supporting transgenic porcine kidney, were included in the present study. Quantitative and qualitative analysis of proteinuria, evaluated with urinary protein to creatinine ratio (UPC ratio) and sodium dodecyl sulphate-agarose gel electrophoresis (SDSAGE) respectively, have been performed. Results: In urine samples, the measurement of UPC ratio was low before transplantation and increased after transplantation. Similarly, SDS-AGE was negative before transplantation but evidenced bands consistent with mixed (i.e. tubular and glomerular) proteinuria in all the samples collected posttransplantation. Proteinuria and presence of LMW proteins was consistently found in urine after transplantation, independently of the fluctuations of creatinine values and/or of the status of renal functions. Conclusions: The evaluation of UPC ratio and the use of SDS-AGE technique in urine samples of cynomolgus monkey recipients of a life-supporting renal xenograft, may be considered a valid, cheap and rapid technique to monitor proteinuria in the post-transplanted period